Method and kit providing bioadhesive binding or coating with polyphenolic mussel proteins

ABSTRACT

The present invention pertains to a method for attaching two surfaces to each other or coating a surface, comprising the steps of providing a bioadhesive composition consisting of a bioadhesive polyphenolic protein derived from a byssus-forming mussel, mixing the bioadhesive protein with a strongly alkaline solution before or simultaneously as applying the composition to the surfaces which are to be attached to each other or the surface to be coated. The surfaces are then joined and left for a sufficiently long time to allow curing to occur alternatively the surface coated by the composition is left for a sufficiently long time to allow curing to occur. The invention can be provided as a kit of parts comprising the bioadhesive protein solution and a preparation of a strongly alkaline solution.

The present invention pertains to a method for attaching two surfaces toeach other or coating a surface, comprising the steps of providing abioadhesive composition consisting of a bioadhesive polyphenolic proteinderived from a byssus-forming mussel, mixing the bioadhesive proteinwith a strongly alkaline solution before or simultaneously as applyingthe composition to the surfaces which are to be attached to each otheror the surface to be coated. The surfaces are then joined and left for asufficiently long time to allow curing to occur alternatively thesurface coated by the composition is left for a sufficiently long timeto allow curing to occur. The invention can be provided as a kit ofparts comprising the bioadhesive protein solution and a preparation of astrongly alkaline solution.

BACKGROUND OF THE INVENTION

Attachment of different structures is crucial in a wide variety ofprocesses. However, this is frequently associated with problems ofdifferent nature depending on what structures are to be attached.

Areas that are particulary troublesome are adhesion in the medicalfield, and attachment of components of very small size, such as in themicro- and nano-techniques. In the medical field, examples of whenadhesives have to be used to adhere biological material include repairof lacerated or otherwise damaged organs, especially broken bones anddetached retinas and corneas. Dental procedures also often requireadhesion of parts to each other, such as during repair of caries,permanent sealants and periodontal surgery. It is very important inbio-medical applications of an adhesive and coating composition to usebioacceptable and biodegradable components, which furthermore should notper se or due to contamination induce any inflammation or toxicreactions. In addition, the adhesive has to be able to attach structuresto each other in a wet environment. In the electronic industry, aparticular problem today is that the components that are to be attachedto each other often are of very small size, and the amount of adhesivethat is possible to use is very small. Adhesives that provide highadhesive strength even with minor amounts of adhesive are thereforerequired. Also for non-medical uses, an adhesive that is non-irritating,non-allergenic, non-toxic and environmentally friendly is preferred, incontrast to what many of the adhesives commonly used today usually are.

Polyphenolic proteins, preferentially isolated from mussels, are knownto act as adhesives. Examples of such proteins can be found in e.g. U.S.Pat. No. 4,585,585. Their wide use as adhesives has been hampered byproblems related to the purification and characterisation of theadhesive proteins in sufficient amounts. Also, mostly when using thepolyphenolic proteins as adhesives the pH has had to be raised toneutral or slightly basic (commonly to from 5.5 to 7.5) in order tofacilitate oxidation and curing of the protein. However, this curing isslow and results in poor adhesive strength and therefore oxidisers,fillers and cross-linking agents are commonly added to decrease thecuring time and obtain a stronger adhesive.

Mussel adhesive protein (MAP) is formed in a gland in the foot ofbyssus-forming mussels, such as the common blue mussel (Mytilus edulis).The molecular weight of MAP from Mytilis edulis is about 130.000 Daltonand it has been disclosed to consist of 75-80 closely related repeatedpeptide sequences. The protein is further characterised by its manyepidermal growth factor like repeats. It has an unusual high proportionof hydroxy-containing amino acids such as hydroxyproline, serine,threonine, tyrosin, and the uncommon amino acid3,4-dihydroxy-L-phenylalanine (Dopa) as well as lysine. It may beisolated either from natural sources or produced biotechnologically.U.S. Pat. No. 5,015,677 as well as U.S. Pat. No. 4,585,585 disclose thatMAP has very strong adhesive properties after oxidation andpolymerisation, e.g. by the activity of the enzyme tyrosinase, or aftertreatment with bifunctional reagents.

MAP is previously known to be useful as an adhesive composition e.g. forophthalmic purposes. Robin et al., Refractive and Corneal Surgery, vol.5, p. 302-306, and Robin et al., Arch. Ophthalmol., vol. 106, p.973-977, both disclose MAP-based adhesives comprising an enzymepolymiser. U.S. Pat. No. 5,015,677 also describes a MAP-based adhesivecontaining a cross-linking agent and optionally a filler substance and asurfactant. Preferred cross-linking agents according to U.S. Pat. No.5,015,677 are enzymatic oxidising agents, such as catechol oxidase andtyrosinase, but sometimes also chemical cross-linking agents, such asglutaraldehyde and formaldehyde can be used. Examples of fillers areproteins, such as casein, collagen and albumin, and polymers comprisingcarbohydrate moieties, such as chitosan and hyaluronan. U.S. Pat. No.5,030,230 also relates to a bioadhesive comprising MAP, mushroomtyrosinase (cross-linker), SDS (sodium dodecyl sulfate, a surfactant)and collagen (filler). The bioadhesive is used to adhere a corneaprosthesis to the eye wall.

EP-A-343 424 describes the use of a mussel adhesive protein to adhere atissue, cell or another nucleic acid containing sample to a substrateduring nucleic acid hybridisation conditions, wherein the musseladhesive protein, despite the harsh conditions encountered during thehybridisation, provided adherence. U.S. Pat. No. 5,817,470 describes theuse of mussel adhesive protein to immobilise a ligand to a solid supportfor enzyme-linked immnoassay. Mussel adhesive protein has also been usedin cosmetic compositions to enhance adherence to nails and skin (WO88/05654).

A major problem associated with known MAP-based bioadhesivecompositions, despite the superior properties of MAP per se, is thatsome constituents, in particular the presently used cross-linkingagents, can harm and/or irritate living tissue and cause toxic andimmunological reactions. Chemical crosslinking agents, such asglutaraldehyde and formaldehyde, are generally toxic to humans andanimals, and it is highly inappropriate to add such agents to asensitive tissue, such as the eye. Enzymes, such as catechol oxidase andtyrosinase, are proteins, and proteins are generally recognised aspotential allergens, especially in case they originate from a speciesother than the patient. Because of their oxidising and hydrolysingabilities, they can also harm sensitive tissue.

Therefore, there is still a need for new adhesive compositions, both formedical and other applications, that provide strong adhesion with smallamounts of adhesive, that are simple to use and that do not cause toxicand allergic reactions.

SUMMARY OF THE INVENTION

The present invention pertains to a method for attaching two surfaces toeach other or coating a surface, comprising the steps of providing abioadhesive composition consisting of a bioadhesive polyphenolic proteinderived from a byssus-forming mussel, mixing the bioadhesive proteinwith a strongly alkaline solution before applying the composition to thesurfaces which are to be attached to each other or the surface to becoated. The surfaces are then joined and left for a sufficiently longtime to allow curing to occur or the surface coated is left to cure fora sufficiently long time. The invention can be provided as a kit ofparts comprising the bioadhesive protein solution and a preparation of astrongly alkaline solution. Since the provided compositions arenon-toxic and presumably non-allergenic the invention is especiallysuitable for use in medical applications for adherence or coating inbiological tissues. Also, since very strong adhesive strengths areprovided using the compositions of the present invention, it is alsoparticularly useful for applications where only minute amounts ofadhesives can be used, including non-biological surfaces.

Definitions

As disclosed herein, the terms “polyphenolic protein”, “mussel adhesiveprotein” or “MAP” relates to a bioadhesive protein derived frombyssus-forming mussels. Examples of such mussels are mussels of thegenera Mytilus, Geukensia, Aulacomya, Phragmatopoma, Dreissenia andBrachiodontes. Suitable proteins have been disclosed in a plurality ofpublications, e.g. U.S. Pat. No. 5,015,677, U.S. Pat. No. 5,242,808,U.S. Pat. No. 4,585,585, U.S. Pat. No. 5,202,236, U.S. Pat. No.5,149,657, U.S. Pat. 5,410,023, WO 97/34016, and U.S. Pat. No.5,574,134, Vreeland et al., J. Physiol., 34: 1-8, and Yu et al.,Macromolecules, 31: 4739-4745. They comprise about 30-300 amino acidresidues and essentially consist of tandemly linked peptide unitsoptionally separated by a junction sequence of 0-10 amino acids. Acharacteristic feature of such proteins is a comparatively high amountof positively charged lysine residues, and in particular the unusualamino acid DOPA (L-3,4-dihydroxyphenylalanine). A polyphenolic proteinsuitable for use in the present invention has an amino acid sequence inwhich at least 3% and preferably 6-30% of the amino acid residues areDOPA. A few examples of typical peptide units are given below. However,it is important to note that the amino acid sequences of these proteinsare variable and that the scope of the present invention is not limitedto the exemplified subsequences below as the skilled person realisesthat bioadhesive polyphenolic proteins from different sources can beregarded as equivalent:

-   a) Val-Gly-Gly-DOPA-Gly-DOPA-Gly-Ala-Lys-   b) Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys-   c) Thr-Gly-DOPA-Gly-Pro-Gly-DOPA-Lys-   d) Ala-Gly-DOPA-Gly-Gly-Leu-Lys-   e) Gly-Pro-DOPA-Val-Pro-Asp-Gly-Pro-Tyr-Asp-Lys-   f) Gly-Lys-Pro-Ser-Pro-DOPA-Asp-Pro-Gly-DOPA-Lys-   g) Gly-DOPA-Lys-   h) Thr-Gly-DOPA-Ser-Ala-Gly-DOPA-Lys-   i) Gln-Thr-Gly-DOPA-Val-Pro-Gly-DOPA-Lys-   j) Gln-Thr-Gly-DOPA-Asp-Pro-Gly-Tyr-Lys-   k) Gln-Thr-Gly-DOPA-Leu-Pro-Gly-DOPA-Lys

The term “surface” is to be interpreted broadly and may comprisevirtually any surface. The choice of surface is not critical to thepresent invention. Examples of surfaces for which the invention arespecially suitable for include non-biological surfaces such as glass,plastic, ceramic and metallic surfaces etc., and biological surfaces,comprising wood and different tissues such as skin, bone, teeth, theeye, cartilage, etc.

By “sufficiently long time” is meant a time period long enough to allowcuring of the bioadhesive composition. Typically the time periodrequired is from at least 10 sec to one hour.

By “strongly alkaline solution” is meant an aqueous alkaline solution,the pH of said strongly alkaline solution being 10 or more, preferably11 or more.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to provide an adhesivecomposition to be used for attaching two surfaces to each other orcoating a surface. The compositions provided in the invention can inprinciple be used to attach any surfaces to each other or to coat anysurface. However, the compositions according to the present inventionare particularly useful when adhesive or coating compositions are neededthat are non-toxic, non-irritating or non-allergenic, or that can beused in wet environments. Also the compositions of the present inventionare useful when a strong adhesion even with small amounts of adhesive,are required. Further advantages with the compositions provided in thepresent invention are their water solubility, the avoidance of organicsolvents commonly used in adhesive or coating compositions, that theyare biologically produced and harmless to the environment.

The only mandatory components of the present invention is thepolyphenolic protein and an alkaline solution. Previously whenpolyphenolic proteins have been used, it has been thought to benecessary to add additional components, such as fillers and oxidisingagents, in order to achieve strong enough adhesive strength and the pHis commonly raised to neutral or slightly basic. The present inventorhas shown that a very strong adhesion, comparable to the adhesivestrength provided using the commonly used MAP compositions, can beprovided simply using a solution of the MAP protein and raising the pHwhen using the composition, employing a very strongly alkaline solutionwith a pH of 10 or more, preferably of 11 or more. Preferred bases forthe present invention are bases such as NaOH, KOH, NH₃ and Na₂CO₃.

Preferably, the MAP concentration of the present invention is above 10mg/ml. More preferably the concentration of the MAP-solution is above 20mg/ml. Typically the concentration is between 20 and 50 mg/ml.

One preferred object of the present invention is to provide an adhesiveor coating composition for medical applications, e.g. for attachingbiological and/or non-biological components to biological structures, anobject for which the MAP protein in itself is well suited, since it isnon-toxic and biodegradable. However, the components commonly added toMAP compositions in order to obtain cross-linking and oxidation(chemical and/or enzymatic crosslinkers and oxidising agents) of thecomposition can lead to irritation and allergic reactions and those MAPcompositions are therefore not optimal for medical applications. Due tothe lack of such components in the present invention, the compositionsof the present invention are particularly suitable for attachment ofbiological surfaces to each other or to biological or non-biologicalcomponents. For the above reasons the compositions of the presentinvention are also particularly useful for coating of materials used inmedical applications or biological tissues.

Due to the very high adhesive strength provided with very small amountsof the compositions of the present invention, one preferred field ofapplication for which the compositions are particularly suitable forattachment of non-biological surfaces such as glass, plastic, ceramicand metallic surfaces. This is particularly useful within the electronicmicro- and nano-techniques, optics, etc. for adhesion or coating of, forexample, biosensors, microchips, solar cells, mobile phones, etc., sincefor these applications only minute amounts of adhesive can be used. Thecompositions of the present invention are also suitable for coating ofnon-biological surfaces.

The adhesive compositions of the present invention are also useful forattachment of cells, enzymes, antibodies and other biological specimento surfaces.

According to one aspect of the invention the solution of MAP is mixedwith a strongly alkaline solution with a pH of 10 or more, preferably 11or more. The mixture is then applied to at least one of the surfaces tobe attached to each other or to the surface to be coated. Alternatively,the MAP-solution and the strongly alkaline solution are separatelyapplied, without any specific order, to at least one of the surfaces,which are to be attached to each other, or a surface to be coated. TheMAP-solution can also be applied to one of the surfaces that are to beattached to each other while the strongly alkaline solution is appliedto the other. If two surfaces are to be attached to each other they arethen joined. Finally the attached or coated surfaces are left for asufficiently long time to allow curing. The time necessary for curingwill for example depend on the surfaces attached or coated, and theamount and the composition of the adhesive.

The present invention is preferably provided as a kit of parts useful ina method for attaching surfaces to each other or coating surfaces,comprising the MAP-solution, a preparation of the strongly alkalinesolution and optionally at least one device, such as a syringe, to applythe compositions to the surfaces that are to be attached or coated.Preferred pH, concentration ranges of the MAP-solution, curing times andsurfaces for use of this kit are as described above.

EXAMPLE 1

Determination of Adhesive Strength for Adhesion between Glass andBiological Tissue with Under Wet Conditions

In order to determine the adhesive strength using the compositions ofthe present invention, the adhesive strength between glass plates andbiological tissue (muscle from cattle and pig) was determined. TheMAP-solution (in 0.01 M citric acid, from Biopolymer Products of SwedenAB. Alingsås, Sweden) of varying concentration and volumes (see Table 1and 2) was applied to a glass plate (75×25×2 mm). Thereafter thestrongly alkaline solution (NaOH, see Table 1 and 2) was applied andcarefully mixed with the MAP-solution on the glass plate before thebiological tissue (approximately of the size 40×15×4 mm) was placed onthe glass plate and fixed with a clip. The pH of the NaOH solutionsemployed were: 1M NaOH pH 14 and 0.1 M NaOH pH 12.5. The samples wereallowed to cure under water (35° C. for 10 min [Table 1], or 1 hour[Table 2]). The adhered surface between the glass plate and thebiological tissue was in most cases 0.2-0.4 cm², with a variation from0.1 to 0.8.

To measure the adhesive strength, the clip was removed from the sampleand the sample was attached to a spring balance via the glass plate. Thebiological tissue was then pulled until it detached from the glass plateand the force needed for this was determined (Table 1 and 2).

Control samples were prepared as described above but instead of analkaline solution an oxidant (NaIO₄, see Table 1 and 2) was mixed withthe MAP-solution before joining the two surfaces.

As can be seen from Table 1 and 2, the adhesive strength employing thecompositions of the present invention (MAP together with a stronglyalkaline solution), adhesive strengths similar or above what could beachieved using an oxidant were unexpectedly obtained.

EXAMPLE 2

Determination of Adhesive Strength for Adhesion between BiologicalTissues with Curing Under Wet Conditions

In order to determine the adhesive strength using the compositions ofthe present invention, the adhesive strength between two attachedbiological tissues (muscle from cattle and pig) was determined. TheMAP-solution (in 0.01 M citric acid, from Biopolymer Products of SwedenAB, Alingsås, Sweden) of varying concentration and volumes (see Table 3)was applied to one of the surfaces that were to be attached to eachother. Thereafter the strongly alkaline solution (NaOH, see Table 3) wasapplied and carefully mixed with the MAP-solution before joining the twosurfaces and fixing them with a clip. The pH of the NaOH solutionemployed was 12.5. The sample was thereafter placed under water (35° C.for one hour) for curing to occur. The adherence surfaces were in mostcases 0.2-0.4 cm², with a variation from 0.1 to 0.8. TABLE 1 Adhesivestrength between glass plates and biological tissue with curing at 35°C. under water for 10 min. MAP MAP NaOH NaOH NaIO₄ NaIO₄ AdhesiveConcentration Amount Concentration Amount Concentration Amount strengthSample (mg/ml) (μg) (M) (μl) (M) (μl) (g) 1 28 84 1.0 2 — — 130 2 27 811.0 2 — — 110 3 28 84 — — 0.01 2 70

TABLE 2 Adhesive strength between glass plates and biological tissuewith curing at 35° C. under water for 1 hour. MAP MAP NaOH NaOH NaIO₄NaIO₄ Adhesive Concentration Amount Concentration Amount ConcentrationAmount strength Sample (mg/ml) (μg) (M) (μl) (M) (μl) (g) 1 23 69 1.0 2— — 100 2 23 69 1.0 2 — — 150 3 25 75 1.0 2 — — 100 4 23 69 0.1 2 — —120 5 23 69 — — 0.01 2 110

TABLE 3 Adhesive strength between biological tissues with curing at 35°C. under water for 1 hour. MAP MAP NaOH NaOH NaIO₄ NaIO₄ AdhesiveConcentration Amount Concentration Amount Concentration Amount strengthSample (mg/ml) (μg) (M) (μl) (M) (μl) (g) 1 18 54 0.1 1.5 — — 190 2 1854 — — 0.01 1.5 150

To measure the adhesive strength, the clip was removed from the sampleand one of the two attached surfaces was attached to a spring balance.The other surface was then pulled until detachment occurred and theforce needed for this was determined (see Table 3).

Control samples were prepared as described above but instead of analkaline solution an oxidant (NaIO_(4,) see Table 3) was mixed with theMAP-solution before joining the two surfaces.

As can be seen from Table 3, the adhesive strength employing thecompositions of the present invention (MAP together with a stronglyalkaline solution), adhesive strength above what could be achieved usingan oxidant was unexpectedly obtained.

The biological tissues has an inherent property for adherence to eachother. The values given in Table 3 are colTected for this effect.

EXAMPLE 3

Determination of Adhesive Strength for Adhesion between Glass andBiological Tissue with Curing Under Dry Conditions

In order to determine the adhesive strength using the compositions ofthe present invention with curing under dry conditions for short timeperiods, the adhesive strength between attached glass plates andbiological tissue (muscle from cattle and pig) was determined. TheMAP-solution (in 0.01 M citric acid, from Bio-polymer Products of SwedenAB, Alingsås, Sweden) (see Table 4) was applied to a glass plate(75×25×2 mm) before the strongly alkaline solution of varyingconcentrations (NaOH, see Table 4) was applied and carefully mixed withthe MAP-solution on the glass plate. The pH of the NaOH solutionsemployed were: 1M NaOH pH 14 and 0.1 M pH 12.5. Thereafter, thebiological tissue (approximately of the size 40×15×4 mm) was placed onthe glass plate and fixed with a clip. The sample was allowed to curefor 1 min at room temperature. The adhered surface between the glassplate and the biological tissue was in most cases 0.3-0.4 cm².

To measure the adhesive strength, the clip was removed from the sampleand the sample was attached to a spring balance via the glass plate. Thebiological tissue was then pulled until it detached from the glass plateand the force needed for this was determined (see Table 4).

The adhesive strength employing Na₂CO₃ as a strongly alkaline base werealso performed in an identical way as described above (Table 4). The pHof the 1M Na₂CO₃ was 11.5.

EXAMPLE 4

Determination of Adhesive Strength for Adhesion between Non-biologicalMaterials

In order to determine the adhesive properties of the compositions of thepresent invention when used for attachment of non-biological materials,the adhesive strength obtained between two glass plates was determined.The MAP-solution (in 0.01 M citric acid, from Biopolymer Products ofSweden AB, Alingsås, Sweden) (see Table 5) was applied to a glass plate(75×25×2 mm) before the strongly alkaline solution (1.0 M NaOH, pH 14)was applied and carefully mixed with the MAP-solution on the glassplate. Thereafter a second glass plate was placed onto the first glassplate with the adhesive composition and fixed with a clip. Theoverlapping surface between the glass plates was ca 2.5 to 3.0 cm², andthe adhered surface was 0.4-0.5 cm². The glass plates were left to cureat room temperature for 72 hours, before determination of shearstrength. The grip length was 75 mm and the cross head speed was 3mm/min during determination of shear strength. For comparison theadhesive strength between glass plates employing common epoxy adhesive(Bostic AB, Helsingborg, Sweden) (10 mg) was determined. The epoxyadhesive covered a surface of 0.7-0.8 cm².

The adhesive strength obtained using the compositions the presentinvention resulted in very strong adhesive strengths, that can becompared to the adhesive strengths obtained employing ca 250 times moreof a common epoxy glue (see Table 5). Therefore very high adhesivestrengths can be obtained with very small amounts of adhesive when usingthe compositions of the present invention. TABLE 4 Adhesive strengthbetween glass plates and biological tissue with curing in dryenvironment at room temperature for 1 min. NaOH or NaOH or MAP Na₂CO₃(only Na₂CO₃ Con- MAP sample 6) (only Adhesive centration AmountConcentration sample 6) strength Sample (mg/ml) (μg) (M) Amount (μl) (g)1 24 60 3 3 50 2 24 60 3 3 50 3 24 60 1 3 40 4 24 60 1 3 60 5 24 60 0.13 25 6 24 48 1.0 3 100

TABLE 5 Adhesive strength between non-biological materials with curingin a dry environment at room temperature for 72 hours. MAP NaOH EpxoyCon- MAP Con- NaOH glue Adhesive centration Amount centration AmountAmount strength Sample (mg/ml) (μg) (M) (μl) (mg) (N) 1 20 40 1.01 >330(glass plates broke) 2 20 40 1.0 1 100 3 — — — — 10 380

1. Method for attaching two surfaces to each other comprising the stepsof a) providing a bioadhesive composition consisting of an aqueoussolution of a bioadhesive polyphenolic protein derived from abyssus-forming mussel, which protein comprises 30-300 amino acids andconsists essentially of tandemly linked peptide repeats comprising 3-15amino acid residues, wherein at least 3% and preferably 6-30% of theamino acid residues of said bioadhesive polyphenolic protein are DOPA,wherein the concentration of said bioadhesive polyphenolic protein insaid bioadhesive composition is within the range of 10-50 mg/ml; b)providing a strongly alkaline solution with a pH of 10 or more; c) (i)mixing said composition and said strongly alkaline solution and applyingthe mixture to at least one of two surfaces to be attached to each otheror (ii) applying said composition and said strongly alkaline solutionsequentially, without any specific order, to at least one of twosurfaces to be attached to each other, thereby mixing the bioadhesivecomposition and the strongly alkaline solution; d) joining said surfacesto each other; and e) leaving said surfaces for sufficiently long timefor curing to occur.
 2. Method for coating a surface comprising thesteps of a) providing a bioadhesive composition consisting of an aqueoussolution of a bioadhesive polyphenolic protein derived from abyssus-forming mussel, which protein comprises 30-300 amino acids andconsists essentially of tandemly linked peptide repeats comprising 3-15amino acid residues, wherein at least 3% and preferably 6-30% of theamino acid residues of said bioadhesive polyphenolic protein are DOPA,wherein the concentration of said bioadhesive polyphenolic protein insaid bioadhesive composition is within the range of 10-50 mg/ml; b)providing a strongly alkaline solution with a pH of 10 or more; c) (i)mixing said composition and said strongly alkaline solution and applyingthe mixture to the surface to be coated or (ii) applying saidcomposition and said strongly alkaline solution sequentially, withoutany specific order, to the surface to be coated, thereby mixing thebioadhesive composition and the strongly alkaline solution; d) leavingsaid surface for sufficiently long time for curing to occur.
 3. Methodaccording to claim 1, wherein the strongly alkaline solution has a pH of11 or more.
 4. Method according to claim 1, wherein the concentration ofthe bioadhesive polyphenolic protein in the bioadhesive composition isin the range of 10-50 mg/ml.
 5. Method according to claim 1, wherein atleast one of the surfaces to be attached or the surface to be coated isa biological surface.
 6. Method according to claim 1, wherein at leastone of the surfaces to be attached or the surface to be coated is anon-biological surface.
 7. Kit for attaching two surfaces to each otheror coating a surface comprising providing a) a composition consisting ofan (acidic) aqueous solution of a bioadhesive polyphenolic proteinderived from a byssus-forming mussel, which protein comprises 30-300amino acids and consists essentially of tandemly linked peptide repeatscomprising 3-15 amino acid residues, wherein at least 3% and preferably6-25% of the amino acid residues of said bioadhesive polyphenolicprotein are DOPA and wherein the concentration of the bioadhesivepolyphenolic protein is within the range of 10-50 mg/ml; and b) apreparation of a strongly alkaline solution with a pH of 10 or more. 8.Kit according to claim 7 wherein the pH of the strongly alkalinesolution is 11 or more.
 9. Kit according to claim 7 further comprising adevice(s) for applying a specified amount of the solution of thebioadhesive protein and the strongly alkaline solution to at least oneof the surfaces that are to be attached to each other or to the surfacethat is to be coated.